Anti mmp3

The paraffin tissue sections of the ankle samples were placed in a 60°C constant temperature incubator and incubated for 120 min. The sections were dehydrated in an ethanol series with a gradient concentration, followed by deparaffinization in xylene. The sections were immersed in antigen retrieval solution and boiled for 20 min, followed by thrice-washing in PBS. After air-drying the section, goat blocking serum was added dropwise, and the primary antibodies against corresponding proteins were then added dropwise. The sections were incubated overnight in the dark at 4°C. On the next day, after washing the sections with PBS, secondary antibodies were added dropwise, and the sections were incubated at room temperature for 20 min. DAB chromogen solution (ZSGB-bio, China) was added, and the tissue sections were re-stained with hematoxylin for 3 min. The sections were then rinsed thoroughly with tap water before mounting with coverslips. Immunohistochemical staining sections were observed and photographed using an image analysis system (NIS Elements). ImageProPlus6.0 software was used for semi-quantitative analysis of positively stained areas in ankle joint tissues and the IOD/area values were calculated.
The concentration configurations of antibodies against corresponding proteins were as follows: anti-MMP-3 (1:50; Arigo, Taiwan); and anti-RANKL (1:100; Arigo, Taiwan).